Lean 

et al. BMC Pregnancy Childbirth (2021) 21:706

https://doi.org/10.1186/s12884-021-04178-6

RESEARCH Open Access

A prospective cohort study providing

insights for markers of adverse pregnancy
outcome in older mothers
Samantha C. Lean1, Rebecca L. Jones1, Stephen A. Roberts2 and Alexander E. P. Heazell1* 

Abstract 
Background:  Advanced maternal age (≥35 years) is associated with increased rates of adverse pregnancy outcome.
Better understanding of underlying pathophysiological processes may improve identification of older mothers who
are at greatest risk. This study aimed to investigate changes in oxidative stress and inflammation in older women and
identify clinical and biochemical predictors of adverse pregnancy outcome in older women.
Methods:  The Manchester Advanced Maternal Age Study (MAMAS) was a multicentre, observational, prospective
cohort study of 528 mothers. Participants were divided into three age groups for comparison 20–30 years (n = 154),
35–39 years (n = 222) and ≥ 40 years (n = 152). Demographic and medical data were collected along with maternal
blood samples at 28 and 36 weeks’ gestation. Multivariable analysis was conducted to identify variables associated
with adverse outcome, defined as one or more of: small for gestational age (< 10th centile), FGR (<5th centile), still-
birth, NICU admission, preterm birth < 37 weeks’ gestation or Apgar score < 7 at 5 min. Biomarkers of inflammation,
oxidative stress and placental dysfunction were quantified in maternal serum. Univariate and multivariable logistic
regression was used to identify associations with adverse fetal outcome.
Results:  Maternal smoking was associated with adverse outcome irrespective of maternal age (Adjusted Odds Ratio
(AOR) 4.22, 95% Confidence Interval (95%CI) 1.83, 9.75), whereas multiparity reduced the odds (AOR 0.54, 95% CI 0.33,
0.89). In uncomplicated pregnancies in older women, lower circulating anti-inflammatory IL-10, IL-RA and increased
antioxidant capacity (TAC) were seen. In older mothers with adverse outcome, TAC and oxidative stress markers
were increased and levels of maternal circulating placental hormones (hPL, PlGF and sFlt-1) were reduced (p < 0.05).
However, these biomarkers only had modest predictive accuracy, with the largest area under the receiver operator
characteristic (AUROC) of 0.74 for placental growth factor followed by TAC (AUROC = 0.69).
Conclusions:  This study identified alterations in circulating inflammatory and oxidative stress markers in older
women with adverse outcome providing preliminary evidence of mechanistic links. Further, larger studies are
required to determine if these markers can be developed into a predictive model of an individual older woman’s risk
of adverse pregnancy outcome, enabling a reduction in stillbirth rates whilst minimising unnecessary intervention.
Keywords:  Aging, Biomarkers, Hormones, Inflammation, Oxidative stress, Stillbirth, Placental dysfunction

*Correspondence: alexander.heazell@manchester.ac.uk
1
Maternal and Fetal Health Research Centre, Division of Developmental
Biology and Medicine, Faculty of Biology, Medicine and Health, University
of Manchester, St. Mary’s Hospital, 5th Floor (Research), Oxford Road,
Manchester M13 9WL, UK
Full list of author information is available at the end of the article

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Lean et al. BMC Pregnancy Childbirth (2021) 21:706
Introduction
Advanced maternal age (≥35 years) is a growing trend
in high income countries [1, 2]. Large epidemiological
studies and subsequent meta-analysis have identified
maternal age ≥ 35 years as an independent risk factor for
adverse fetal outcomes including: fetal growth restriction
(FGR), pre-term birth (PTB), pre-eclampsia (PE), neona-
tal intensive care unit (NICU) admission and stillbirth
[3–8]. Stillbirths in older women are likely to occur near
term with risks comparable to those in obesity, smok-
ing, diabetes or history of stillbirth [9–11]. However,
unlike these conditions there are few guidelines to reduce
adverse outcomes in older women [12, 13].
There is international recognition that older women
should undergo additional antepartum screening or
intervention to address the increased risk of stillbirth
[14–17]. The RCOG and SOGC recommend offering
induction of labour (IOL) at 39 weeks and/or additional
monitoring from 38 weeks’ gestation [2, 15, 18]. Although
not associated with an increase in the rate of Caesarean
section [19], IOL may be viewed as an unnecessary inter-
vention as the majority of mothers will have uncompli-
cated pregnancies. Furthermore, induction may not be an
acceptable intervention for older women without further
indication, with poor recruitment (only 13.6%) of eligible
women to the 35–39 trial consenting to be randomised
[19]. Identification of mothers with highest risk would
result in fewer interventions to prevent stillbirths.
Many pregnancy pathologies are associated with
changes in oxidative stress and inflammatory status
[20–22]. Similar changes are reported in aging pro-
cesses although these are usually researched in older
populations [23, 24]. If these alterations were present in
older women they could adversely affect placental func-
tion [25–27]. Previous work found evidence of placental
dysfunction in pregnancies in older women including
(but not limited to) reduced amino acid transport, aber-
rant cell turnover and reduced placental efficiency [28].
Therefore, it was hypothesised that the increased risk of
adverse pregnancy outcome results from an aging mater-
nal environment and that a combination of biomarkers
of aging, placental dysfunction, and clinical risk factors
might identify women at greatest risk. This study aimed
to determine whether there were changes in oxidative
stress and inflammation in pregnancies in older mothers
and whether changes in these biomarkers were evident in
adverse pregnancy outcomes in this population.
Methods
Women were recruited to the Manchester Advanced
Maternal Age Study (MAMAS) from March 2012–Octo-
ber 2014 from six UK maternity units after providing
written informed consent. Ethical approval was obtained
Page 2 of 17
from the NRES Committee North West, (12/NW/0015).
Pregnant women aged between 20 and 30 years (controls
– optimal reproductive age), 35–39 years and ≥ 40 years
were approached at 28 weeks’ gestation between April
2012–June 2014. Women with multiple pregnancy, body
mass index (BMI) < 18.5 or > 30 kg/m2, fetal abnormali-
ties, and pre-existing maternal medical conditions were
excluded.
In addition to usual antenatal care, participants
attended prenatal research appointments at 28 and
36 weeks’ gestation (±1 week), at which detailed demo-
graphic, medical data and maternal blood samples for
plasma and serum fractionation were collected. After
delivery, outcome data were collected from medical
records. Biochemical analyses were conducted after
delivery, therefore not altering participants’ prenatal care.
The Index of Multiple Deprivation (IMD) – a measure
of relative social deprivation [29] - was calculated from
the mother’s address using NPEU-IMD tool (University
of Oxford, UK). A composite adverse pregnancy out-
come was defined as one or more: small for gestational
age (SGA) or FGR (< 10th/<5th centile respectively using
individualised birthweight centiles (IBC) [30], still-
birth, admission to the NICU, PTB without infection
(< 37 weeks gestation), and 5 min Apgar score < 7 in the
absence of maternal diseases (diabetes/hypertension).
Normal pregnancy outcome was defined as a term live
birth (38–42 weeks), appropriately grown (IBC between
10-95th centile) and absence of maternal or fetal compli-
cation (not limited just to those included in our defini-
tion of adverse pregnancy outcome). We conservatively
estimated a 20% incidence of adverse pregnancy outcome
(using data from Reference [8] stillbirth rate 0.4%, inci-
dence of FGR 6%, neonatal unit admission 8%, preterm
birth 10%), therefore approximately 600 participants were
required to obtain 120 women with composite adverse
outcome; 120 women with adverse outcome would allow
multivariable analysis of six covariates.
Nested case control studies
Two nested case-control studies were conducted i) to
determine whether pregnancy in older mothers associ-
ated with elevated circulating biomarkers of inflamma-
tion and oxidative stress and ii) to determine whether
adverse pregnancy outcome was associated with markers
of aging and placental dysfunction in women ≥35 years
of age. In the first nested case-control study samples
from participants ≥40  years were matched (1:1) to
mothers aged 20–30 and 35–39 years (n  = 40/group)
for demographic (BMI, IMD, ethnicity (groups), mari-
tal status (married, single, partnership), smoking status
(current/ex/non-smoker) and obstetric characteristics
(normal vaginal delivery (NVD)). Women with adverse
 Lean et al. BMC Pregnancy Childbirth (2021) 21:706
pregnancy outcome, who conceived via assisted repro-
ductive technology (ART) or in whom maternal disease
that developed post 28 weeks’ gestation were excluded.
Sample sizes were determined following power calcula-
tions based on previous studies for detecting a difference
in circulating oxidative stress [31–34] and inflammatory
mediators [35–37]. For example, to detect a difference in
cytokines TNF-α and IL-6 between normal and adverse
outcomes in older women with 80% power with a 5% sig-
nificance level required between 28 and 43 participants
in each group.
For the second nested case control study, cases were 43
women ≥35 years of age who had an adverse outcome as
defined above. Controls were participants with maternal
age ≥ 35 years with a normal pregnancy outcome. Par-
ticipants that had maternal disease that developed post
28 weeks, PTB associated with infection, or incomplete
outcome data were excluded. Groups were matched for
demographic characteristics (maternal/paternal age, eth-
nicity, BMI, smoking status, marital and housing (owner-
ship/rental) status and parity (primi/multiparous.
Biomarkers of aging were measured in maternal serum
or plasma samples for participants included in the nested
case control studies (Supplementary Table  1). Absorb-
ances were measured using a microplate reader (FLU-
OStarOmega, BMG Labtech) for all Enzyme linked
immunosorbent assays (ELISAs). Pro/anti-inflammatory
cytokines (interleukin (IL)-1α, IL-1β, IL-1Ra, IL-6, IL-10
maternal serum using DuoSet®ELISAs (R&D Systems,
and tumour necrosis factor (TNF)-α) were quantified in
Abingdon, UK) following an optimised protocol [27].
tified using OxiSelect™ Total Antioxidant Capacity
Maternal plasma antioxidant concentration was quan-
Assay Kit (Cell Biolabs, Inc., San Diego, USA). Oxida-
tive damage markers 8-Isoprostane and DNA/RNA oxi-
dative damage were measured by EIA Kits and Protein
Carbonyl Calorimetric Assay Kit (Cayman Chemical
Company, Michigan, USA). Maternal serum placental
hormones (hCG, PAPP-A, Progesterone and hPL were
quantified using DRG ELISA kits (DRG Instruments,
Marburg, Germany). Placental growth factor (PlGF) and
using DuoSet® ELISAs (R&D systems). All assays were
soluble fms-like tyrosine kinase-1 (sFlt) were quantified
conducted according to the manufacturer’s standard
protocols.
Statistical analysis
Demographic data were compared using Fisher’s Exact
test for categorical data and Kruskal-Wallis tests with
Dunn’s multiple comparisons or Mann Whitney U tests
for continuous data. Univariate logistic regression was
conducted on the whole dataset to identify demographic
Page 3 of 17
or clinical variables associated with composite adverse
pregnancy outcome. Multivariable logistic regression was
used to quantify the effect of maternal age on adverse
outcome; in this analysis maternal ethnicity, use of ART,
smoking status, parity, IMD and home ownership were
included as categorical variables and in an second model,
paternal age was included as an additional categorical
variable (< 30, 30–34, 35–39 and 40+ years). The logistic
regression analyses were conducted using STATA (Ver-
sion 14, StataCorp, Texas, USA). Biomarker analysis was
performed on GraphPad Prism (Version 6.04, GraphPad
Software Inc., La Jolla, USA) using Kruskal-Wallis with
Dunn’s multiple comparisons tests on untransformed
data or Mann-Whitney U where appropriate. Due to
wide dispersion in the cytokine data, including values
at the lower limit of detection of the assay, these data
were transformed as log(y + smallest detectable value)
for analysis. Gestational age effects were assessed using
Spearman’s correlation. Markers that showed statisti-
cal significance at the p < 0.01 level were analysed to test
their predictive potential as markers of adverse preg-
nancy outcome in women aged ≥35 years by calculation
of the area under the receiver operator characteristic
curve (AUROC).
Results
Overall, 1134 women were approached to participate in
MAMAS. 571 of these mothers (51%) either did not meet
the inclusion criteria or declined to participate (Fig.  1).
563 mothers consented before 28 weeks’ gestation. A fur-
ther 45 participants were recruited between 28 weeks’
gestation and birth. 80 mothers (14%) were either lost to
follow up or withdrew from the study between 28 weeks’
gestation and birth. Therefore, demographic, medi-
cal and pregnancy data was collected on a final cohort
of 528 participants (n  = 154 20–30, n  = 222 35–39 and
n = 152 ≥ 40 year olds).
There were no differences between ethnicity, employ-
ment and deprivation between participants in differ-
ent age groups (Table  1). Paternal age was higher in
pregnancies to women aged 35–39 and ≥ 40 years, and
women in both these groups had higher BMI compared
to 20–30 year olds. More mothers aged 35–39 years were
married, and more mothers aged 20–30 years were in
partnerships than the other maternal age groups. More
older mothers were non-smokers, homeowners, multipa-
rous and had previous or current fertility treatment (pre-
dominantly in  vitro fertilisation) compared to women
aged 20–30. Of those who conceived using assisted
reproductive technologies, 45% of those ≥40 years used
egg and/or sperm donors, compared to 5% of 35–39 year
olds.
Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 4 of 17

Fig. 1  Flow diagram of participant recruitment and retention in the MAMAS cohort study
 Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 5 of 17

Table 1  Demographic data for total MAMAS participants

Demographics 20–30 Years (n = 154) 35–39 Years (n = 222) ≥40 Years (n = 152) P value Overall P value Multiple Comparisons

Maternal age 26 (20–30) 37 (35–39) 42 (40–49)

Paternal Age a 29 (18–48) 38 (21–50) 43 (24–60) < 0.001 †¶Δ
 < 0.0001
2 a †
BMI (kg/m ) 23.8 (18.7–29.9) 24.1 (18.5–29.5) 24.9 (19.0–29.9) 0.0026 0.10, ¶0.0004, Δ0.008
1 missing value
European ­Ethnicityb 90% (138) 87% (191) 95% (144) 0.08
Marital Status b
†<
  Single 5% (7) 2% (5) 5% (8) 0.001  0.001, ¶0.15, Δ0.08
  Married/CP 45% (69) 66% (146) 55% (83)
  Partner 51% (78) 31% (69) 39% (60)
  Other 0% (0) 1% (1) 1% (1)
Occupation b
  Employed – Full Time 55% (84) 49% (109) 59% (89) 0.51
  Employed – Part Time 26% (40) 32% (72) 26% (40)
  Unemployed 18% (28) 16% (35) 14% (21)
  Unknown 1% (2) 3% (6) 1% (2)
Smoking Status b
  Non-smoker 71% (110) 74% (165) 81% (123) 0.056
  Current 11% (17) 5% (12) 3% (5)
  Ex-smoker 18% (27) 20% (45) 16% (24)
Housing b
†¶
  Owns 45% (70) 73% (163) 84% (127) < 0.001 p < 0.001,Δ0.023
a
IMD 19.09 16.11 15.54 0.25
  Score (2.28–71.95) (1.94–76.09) (1.94–69.63)
4 missing values 7 missing values 13 missing values
Parity b
†
  Primiparous 49% (76) 28% (62) 34% (52) < 0.001  < 0.001, 0.001, Δ0.092
  Parous 51% (86) 72% (160) 66% (100)
Previous APO b,c 22% (17) 32% (51) 22% (22) 0.12
  FGR c 13% (10) 21% (33) 11% (11) 0.09
  Stillbirth c 0% (0) 3% (4) 4% (1) 0.52
Previous ART b < 1% (1) 8% (18) 18% (28) < 0.001 †
0.001, ¶ < 0.001, Δ0.004
ART b
< 1% (1) 9% (21) 14% (22) 0.004 †
0.001, ¶ < 0.001, Δ0.005
  Hormonal < 1% (1) 2% (5) 1% (2)
  IVF 0% (0) 6% (13) 7% (10)
  IVF - Donor 0% (0) < 1%(1) 7% (10)
  IUI 0% (0) 1% (2) 0% (0)
Data are mean (range) or percentage (number). BMI Body mass index, CP Civil partnership, IMD Index of multiple deprivation, APO Adverse pregnancy outcome, ART​
Assisted reproductive therapies, IVF In vitro fertilisation, Donor = egg and/or sperm, IUI Intrauterine insemination. Statistical differences are aKruskal-Wallis with Dunn’s
multiple comparisons or b Fishers exact test. c expressed as percentage of parous women. When overall p > 0.05, multiple comparisons p values are reported († 20–30
vs. 35–39 years, ¶ 20–30 vs ≥40 years, Δ 35–39 vs. ≥40 years). Significant differences are highlighted with bold p values
Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 6 of 17

Table 2  Pregnancy outcome of MAMAS participants

Pregnancy Outcome 20–30 Years (n = 154) 35–39 Years (n = 222) ≥40 Years (n = 152) P value Overall P Value Multiple
Comparisons

Gestation at Delivery a 40 + 2 39 + 4 39 + 3 < 0.001 †

0.003, ¶ < 0.0001, Δ0.04
Weeks + days (34 + 1–42 + 5) (39 + 6–43 + 1) (30 + 5–42 + 4)
Birthweight (g) a 3416 (1880–4680) 3438 (1300–4900) 3375 (1480–4420) 0.94 –
Individualised Birthweight 38.4 (0.1–98.7) 45.3 (0.0–99.6) 47.7 (0.0–99.4) 0.35 –
Centile a
Induction of Labour b 32% (50) 26% (58) 43% (66) 0.002 †
0.20, ¶0.06, Δ 0.001
Mode of Birth b
†
  Normal Vaginal Delivery 60% (102) 50% (117) 39% (64) < 0.001 0.001,¶ < 0.001, Δ 0.17
  Elective Caesarean Section 13% (12) 25% (47) 25% (35)
  Emergency Caesarean 8% (12) 10% (25) 17% (26)
Section
  Instrumental Vaginal 19% (28) 15% (33) 19% (27)
Delivery
Maternal Disease b
†
  Preeclampsia 5% (8) 1% (2) 3% (4) 0.04 0.01,¶ 0.38, Δ 0.23
  Gestational Diabetes 1% (2) 3% (7) 3% (4) 0.56
Mellitus
NPO b 77% (119) 81% (180) 80% (121) 0.67
APO b 23% (35) 19% (42) 20% (31) 0.69
Components of APO
  Pre-Term Birth 6% (9) 6% (13) 6% (9) 1.00
  Small for gestational Age 10% (15) 11% (25) 9% (14) 0.73
  Fetal Growth Restriction 3% (5) 7% (16) 4% (6) 0.21
  Large for Gestational Age 3% (4) 5% (12) 4% (6) 0.40
  Neonatal Unit Admission c 4% (6) 7% (15) 7% (11) 0.36
†
  Stillbirth 0% (0) 0% (0) 3% (4) 0.007 1.00, ¶0.06, Δ 0.03
Data are mean (range) or percentage (number). Statistical differences are aKruskal-Wallis with Dunn’s multiple comparisons or b Fishers exact test. c Expressed
as proportion of babies born alive. When overall p > 0.05, multiple comparisons p values are reported († 20–30 vs. 35–39 years, ¶ 20–30 vs ≥40 years, Δ 35–39 vs.
≥40 years)

Older women delivered moderately earlier than women
aged 20–30 years (39 weeks + 3 days vs. 40 weeks + 2 days;
Table 2). Women ≥40 years had a 43% rate of IOL com-
pared to 26–32% in the younger groups (p  = 0.002).
Fewer older mothers had NVDs (39% vs 50 and 60%)
p  = 0.001) and more had elective or emergency caesar-
ean sections. Birthweight, IBC and the incidence of preg-
nancy-related maternal disease did not differ between
the maternal age groups. Four women in MAMAS had
­ 8+ 1 and ­40+ 3 weeks gestation), all
a stillbirths (between 3
were ≥ 40 years old (p = 0.007).
Demographic predictors of adverse pregnancy out-
come: Univariate logistic regression demonstrated that
ex- and current smokers had higher odds of adverse
pregnancy outcome than non-smokers (OR 1.96
(95%CI 1.17–3.30) and 3.97 (95%CI 1.92–8.21), p = 0.01
and < 0.001 respectively; Table 3). Multiparity was protec-
tive against adverse pregnancy outcome (OR 0.67 (95%CI
0.44–1.03), p  = 0.05) compared to primiparous women.
In this population, maternal ethnicity did not indepen-
dently affect the risk of adverse pregnancy outcome. ART
had no detectable effect on outcome (OR 0.94 (95%CI
0.44–2.01) p = 0.88). Paternal age had a protective effect
against adverse outcome (OR 0.54 (95%CI 0.31–0.94)
p = − 0.03). Only the associations between adverse preg-
nancy outcome and maternal parity or cigarette smok-
ing remained statistically significant after adjusting for
maternal ethnicity, parity, smoking status, housing, and
paternal age (Table  3). Following adjustment for covari-
ates maternal age ≥ 40 was associated with increased risk
of adverse perinatal outcome (AOR 2.67, 95% CI 1.09–
6.52, p = 0.03).
 Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 7 of 17

Table 3  Unadjusted and adjusted odds ratios for prediction of adverse pregnancy outcome of MAMAS participants
Normal Outcome Adverse Outcome Unadjusted Model 1 Model 2
(n = 420) (N(%)) (n = 108) (N(%))
OR (95% CI) P value AOR (95% CI) P value AOR P value

Ethnicity
  European 373 (89) 101 (94) Reference Reference Reference
  Asian 24 (6) 5 (5) 0.77 (0.29–2.07) 0.49 0.73 (0.25–2.13) 0.57 0.58 (0.18–1.88) 0.37
  Other 23 (5) 2 (2) 0.32 (0.74–1.38) 0.08 0.27 (0.06–1.28) 0.081 0.27 (0.54–1.32) 0.11
Parity
  Nulliparous 143 (34) 47 (44) Reference Reference Reference
  Multiparous 277 (66) 61 (56) 0.67 (0.44–1.03) 0.07 0.58 (0.36–0.93) 0.03 0.56 (0.33–0.89) 0.02
ART​
  None 383 (91) 99 (92) Reference Reference Reference
  ART​ 37 (9) 9 (8) 0.94 (0.44–2.01) 0.88 1.01 (0.44–2.29) 0.99 0.85 (0.35–2.04) 0.71
Smoking
 No 332 (79) 66 (61) Reference Reference Reference
 Ex 69 (16) 27 (25) 1.96 (1.17–3.30) 0.01 1.86 (1.08–3.24) 0.03 1.85 (1.05–3.25) 0.03
 Current 19 (5) 15 (14) 3.97 (1.92–8.21) < 0.001 4.24 (1.88–9.54) < 0.001 4.22 (1.83–9.75) 0.001
Housing
 Rented 123 (29) 45 (42) Reference Reference Reference
 Owns 297 (71) 63 (58) 0.58 (0.37–0.90) 0.01 0.57 (0.34–0.97) 0.04 0.56 (0.32–0.98) 0.04
IMD Quintile
  1st 73 (18) 19 (18) Reference Reference Reference
  2nd 78 (20) 29 (27) 1.42 (0.74–2.74) 0.29 1.31 (0.66–2.60) 0.53 1.37 (0.68–2.76) 0.38
  3rd 97 (24) 13 (12) 0.51 (0.24–1.11) 0.09 0.46 (0.21–101) 0.05 0.48 (0.21–1.10) 0.08
  4th 68 (17) 21 (20) 1.19 (0.59–2.40) 0.63 0.92 (0.44–1.93) 0.82 0.98 (0.46–2.10) 0.97
  5th 82 (21) 22 missing 14 (23) 2 missing 1.12 (0.57–2.22) 0.74 0.98 (0.46–2.08) 0.96 1.02 (0.48–2.23) 0.94
values values
Paternal Age
  < 30 years 86 (21) 33 (32) Reference Reference
  30–34 years 58 (14) 16 (15) 0.77 (0.39–1.51) 0.45 0.86 0.72
  35–39 years 115 (28) 24 (23) 0.54 (0.30–0.98) 0.04 0.54 0.16
  ≥40 years 148 (36) 31 (90) 0.54 (0.31–0.94) 0.03 0.46 0.08
13 missing values 4 missing values
Model 1 – multivariable logistic regression including all variables except paternal age (ethnicity, parity, smoking, house ownership, IMD quintile and maternal age
group), Model 2 – multivariable regression additionally adjusting for paternal age. ART​Assistive Reproductive Techniques, IMD Index of multiple deprivation, IMD
Quintile 1st = least deprived, 5th = most deprived. Statistics performed were univariate and multivariable meta-regression. Adjustments included maternal ethnicity,
parity, smoking status, housing, and paternal age
Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 8 of 17

Fig. 2  Circulating inflammatory markers in women grouped by maternal age, n = 40/group. Maternal serum at 28 weeks (A, C, E, G, I) and
36 weeks (B, D, F, H, J) gestation concentrations of: A, B interleukin (IL-1) α, C, D IL-1β, E, F IL-1Ra, G, H IL-10 and (I-J) TNFα. Data are logarithmically
transformed, with median, interquartile range (box) and total range (whiskers) plotted. Analysed using one-way ANOVA (*p < 0.05, **p < 0.01)
 Lean et al. BMC Pregnancy Childbirth (2021) 21:706
Nested case‑control study to determine whether advanced
maternal age associated with elevated circulating
biomarkers of inflammation and oxidative stress
The characteristics of participants (n = 40/group) in this
nested study are shown in Supplementary Table  2. The
three groups differed for factors strongly associated with
older age: higher home-ownership in 35–39 and ≥ 40 year
olds (95% of ≥40 vs. 90% of 35–39 vs. 58% of 20–30 years)
and multiparous (78% of ≥40 vs. 80% of 35–39 vs. 53%
of 20–30 yeas) and mothers ≥40 years had higher previ-
ous miscarriage rates (53% vs. 25% of 20–30 years). Older
mothers gave birth earlier (by 5–7 days) than 20–30 year
olds (p < 0.001; Supplementary Table 3).
Large variation was seen in inflammatory biomarker
concentrations in maternal serum. At 28 weeks’ ges-
tation, lower circulating concentrations of IL-1α and
IL-1RA (p  = 0.04 and 0.005, Fig.  2A, E) were meas-
ured in mothers 35–39 years compared to 20–30 years,
but no significant differences were present between
20 and 30 and  40 
≥  year olds. Similar trends were
apparent at 36 weeks gestation but were not statisti-
cally significant. Anti-inflammatory IL-10 was lower
in mothers ≥40 years at 28 weeks’ (p  = 0.05), with a
stepwise age-related decrease apparent at 36  weeks
(p  = 0.03, Figs.  2G-H). No differences were seen in
IL-1β or TNF-α at 28 weeks or 36 weeks between age
groups (Fig.  2). There were no differences in maternal
circulating markers of oxidative stress at 28 weeks’ ges-
tation (Fig.  3A, C, E). TAC was increased at 36 weeks’
gestation in mothers ≥40 years compared to controls
(p  = 0.015; Fig.  3B). No differences of other oxidative
stress markers were seen (Fig.  3D and F, Supplemen-
tary Figure 1). Gestational age affected TAC in mothers
≥40s (p = 0.004), whereas 8-isoprostane was positively
related to gestational age in all groups (p  < 0.001; data
not shown). When assessing change in oxidative sta-
tus over time, TAC levels fell across the third trimester
in controls but increased in older women (p  = 0.005;
Fig.  3G). In contrast, elevated lipid peroxidation
(8-isoprostane) was apparent in mothers 20–30 and
35–39 years across the third trimester but decreased
in women ≥40 (p  = 0.04; Fig.  3H). There was a posi-
tive relationship between TAC and 8-isoprostane in all
participants at 28 weeks’ gestation, strongest in women
35–39 years (r  = 0.61 vs 0.45 in women aged 20–30,
(See figure on next page.)
Fig. 3  Levels of markers of oxidative stress status in women grouped by maternal age; n = 40/group. Maternal serum at 28 weeks (A, C, E) and
36 weeks (B, D, F) gestation was quantified for: A, B Total Antioxidant Capacity (TAC), C, D 8-Isoprostane and E, F protein Carbonyl. Rate of change
between 28 and 36 weeks’ gestation of: G Total Antioxidant Capacity (TAC) and H 8-Isoprostane. Data presented as median, interquartile range
(box) and total range (whiskers). Analysed using Kruskal-Wallis with Dunn’s multiple comparisons or one-way ANOVA on transformed data. TAC was
correlated with log 8-isoprostane at I 28 weeks and J 36 weeks using Spearman rank test (*p < 0.05, **p < 0.01), *** p < 0.001)
Page 9 of 17
Fig.  3I). At 36 weeks a negative correlation existed
between TAC and 8-isoprostane in women 35–39
and ≥ 40 years (r  = − 0.42 and − 0.39 respectively;
p = 0.005 and p = 0.01 Fig. 3J), whereas no relationship
was seen in women aged 20–30 (r = − 0.17, p = 0.32).
Nested case‑control study to determine whether maternal
biomarkers associated with adverse pregnancy outcome
in older women
Women ≥35 years of age who had an adverse pregnancy
outcome (n  = 43) were well matched for demographic
variables to women ≥35 years of age who had a normal
pregnancy outcome (Supplementary Table 4). The major-
ity of infants in the group of women with adverse preg-
nancy outcome were classified as SGA (84%), with 44%
under the 5th centile (FGR) (Supplementary Table  5).
There were fewer NVDs (39% vs. 65%, p  = 0.02) and
more EMCS (23% vs. 7%, p  = 0.04) in the group with
adverse pregnancy outcomes compared to those with
a normal outcome. A quarter of mothers in the adverse
pregnancy outcome group delivered preterm (< 37 weeks,
p  < 0.001) and a quarter of infants were admitted to
NICU (p < 0.001). There were three stillbirths included in
the adverse pregnancy outcome group (one stillbirth was
excluded due to congenital abnormality identified as the
cause of death).
There were no differences in circulating cytokines at
28 weeks’ or 36 weeks’ gestation between older women
with normal and adverse pregnancy outcome (Supple-
mentary Figure 2). TAC was higher in older mothers with
adverse pregnancy outcome compared to normal out-
comes at both 28 and 36 weeks’ gestation (p = 0.002 and
0.006 respectively, Fig.  4A, B). 8-isoprostane increased
between 28 and 36 weeks’ gestation in both normal and
adverse pregnancy outcome groups (data not shown).
8-isoprostane also significantly increased in older moth-
ers with adverse pregnancy outcome at 28 weeks but
was not elevated at 36 weeks’ gestation (p  = 0.01 and
p  = 0.82 respectively, Fig.  4E-F). No differences were
detected in markers of DNA/RNA damage or protein
carbonyl between groups at either 28 weeks (p  = 0.66
and 0.34 respectively) or 36 weeks’ gestation (p  = 0.57
and 0.60 respectively; Fig.  4C, D, G, H). A positive cor-
relation between TAC and 8-isoprostane at 28 (r  = 0.46
for adverse pregnancy outcome and 0.31 for normal
Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 10 of 17

Fig. 3  (See legend on previous page.)

 Lean et al. BMC Pregnancy Childbirth (2021) 21:706
pregnancy outcome) and at 36 weeks with normal out-
comes (r = − 0.39; p = 0.01 Fig. 4J), whereas there was no
relationship seen in women with adverse pregnancy out-
come (r = − 0.33, p = 0.07, Fig. 4I-J).
Concentrations of hPL, PlGF and PlGF:sFlt ratio were
unchanged at 28 weeks’ gestation (Fig.  5A, C, G). hPL
was lower at 36 weeks’ gestation in women with adverse
pregnancy outcome (p  = 0.007; Fig.  5B). Similarly, PlGF
concentrations were lower at 36 weeks’ when measured
alone (p  < 0.001, Fig.  5D) or adjusted for sFlt-1 (human
VEGF R1/Flt-1) concentrations (p  = 0.03, Fig.  5H).
sFlt-1 was lower in women with adverse pregnancy out-
come at 28 weeks’ with a similar trend at 36 weeks’ ges-
tation (p = 0.05 and 0.07, Fig. 5E,F). No differences were
detected in circulating hCG, PAPP-A or progesterone
with adverse pregnancy outcome (Supplementary Fig-
ure 3). ROC curves were created for all biomarkers that
reached a statistical significance of p < 0.01 between nor-
mal pregnancies and those with adverse pregnancy out-
come. TAC and 8-isoprostane had predictive area under
the curve values of 0.69 and 0.66 respectively (ranked as
a poor prognostic markers, Fig.  6A-B), whilst hPL and
PlGF had predictive values of 0.68 (poor) and 0.74 (fair),
respectively (Fig. 6C-D).
Discussion
This prospective study found that increased maternal age
was associated with increased total antioxidant capacity
and a reduction in anti-inflammatory IL-10 and IL-RA
indicating changes in oxidative stress and inflammation
over the timeframe of reproductive life span. In mothers
≥35 years of age, adverse pregnancy outcome was associ-
ated with a further increase in total antioxidant capacity
and a reduction in placentally-derived biomarkers, hPL
and PlGF. In this population, maternal smoking and pri-
miparity were independent risk factors for adverse out-
come, consistent with retrospective study findings [7, 10,
38].
Strengths and limitations
This prospective study was designed to explore poten-
tial mechanisms underpinning the association between
advanced maternal age and adverse pregnancy outcome,
to identify potential biomarkers and generate further
hypotheses which can be explored. The multi-centre
(See figure on next page.)
Fig. 4  Levels of oxidative stress markers maternal serum from women ≥ 40 years of age with normal and adverse pregnancy outcome; n = 43/
group. Maternal serum at 28 weeks (A, C, E, G) and 36 weeks (B, D, F, H) gestation against pregnancy outcome. A, B Total Antioxidant Capacity (TAC),
C, D DNA/RNA damage, E, F 8-Isoprostane and G, H Protein Carbonyl. Data are (A, B) median, IQR and range, Mann Whitney test or (C-H) unpaired
t-test for transformed data. TAC was correlated with 8-isoprostane at (G) 28 weeks and (H) 36 weeks gestation using Spearman rank test (*p < 0.05,
**p < 0.01)
Page 11 of 17
approach used in this study offered diversity, making it
more representative of the UK maternity population.
However, this study would have benefitted from larger
overall sample size to increase the statistical power to
confirm associations between adverse pregnancy out-
come and biomarkers and assess the predictive ability
of combinations of clinical and biochemical markers.
Furthermore, a larger cohort would have enabled larger
nested case control studies, with greater statistical power,
particularly for cytokine biomarkers which had wide
variation in levels between individuals. In addition, the
study excluded women with a BMI above 30, which may
account for some of the increased adverse outcomes
observed in older women (as older women have a higher
BMI than younger women). We recognize this limitation
of the study and believe that BMI is an important covari-
ate to include in future studies to determine the degree of
interaction between these two variables that are indepen-
dently associated with adverse pregnancy outcome.
Despite these limitations, MAMAS is a large prospec-
tive study investigating associated factors for adverse
pregnancy outcome in older women and our detailed
data collection provided the ability to adjust for multiple
confounding variables, and delineate the effects of pater-
nal age, ethnicity and parity and socioeconomic status, all
of which may be associated with stillbirth [39–42].
Interpretation
Consistent with other larger retrospective studies the
clinical factors associated with adverse outcome in
older women were maternal smoking and primiparity
[8, 10, 38]. However, in our study population concep-
tion by ART had no significant association with stillbirth,
although the effect-size of other studies was within the
95% confidence intervals of our population [43, 44]. It
is notable that in this and other studies, older mothers
were more likely to own their own homes, be in a stable
relationship, have higher rates of education and had the
lowest rates of cigarette smoking [45], factors which are
associated with lower risk of adverse outcomes such as
stillbirth and fetal growth restriction [46]. However, the
opposite finding that older women have higher rates of
these conditions is noted in observational studies [8].
These findings suggest that other mechanisms much
account for the increased rate of adverse maternal and
Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 12 of 17

Fig. 4  (See legend on previous page.)

 Lean et al. BMC Pregnancy Childbirth (2021) 21:706
neonatal outcomes in older mothers. Furthermore, they
also emphasise the need to promote smoking cessation
services and suggest that women who have their first
pregnancy over the age of 35 or those who smoke should
be prioritised for intervention.
Understanding the mechanisms underlying the suscep-
tibility to adverse outcomes is essential to improve identi-
fication of older women highest risk of adverse outcomes.
This study focussed on processes implicated in maternal
aging and placental dysfunction. A pro-inflammatory
bias and elevated oxidative stress are established hall-
marks of aging [47–49] and both are strongly associated
with pregnancy pathologies, particularly those character-
ised by placental dysfunction [50–52]. The nested case
control studies revealed features of biological aging in
older women in the absence of adverse outcome includ-
ing: elevations in TAC, coincident with reduced oxidative
damage (reduced lipid peroxidation) and a reduction in
anti-inflammatory cytokines (IL-10 and IL-1Ra) possi-
bly indicating a shift towards a pro-inflammatory state.
The former findings are consistent with these women
delivering healthy infants and suggest that adaptive
antioxidant responses are effective in maintaining the
oxidant:antioxidant balance protecting against oxidative
stress [53]. The reduction in IL-10 levels has also been
seen in serum and placenta of women perceiving reduced
fetal movements [27] and in the placenta of infants with
FGR [54]. As both FGR and RFM are associated with pla-
cental dysfunction, the reduction in IL-10 could be con-
sistent with the increased placental dysfunction seen in
older mothers [28]. Critically, an isolated reduction in
anti-inflammatory status is not detrimental, but studies
of the IL-10 knockout mouse demonstrate increased sus-
ceptibility to inflammatory stimuli resulting in PTB and
fetal loss [55], and exacerbation of the vascular symp-
toms of preeclampsia [56] and effects of hypoxia [57].
Therefore, an age-related decline in anti-inflammatory
cytokines may increase vulnerability of older women
to inflammation and the associated detrimental effects
observed on placental function and should therefore be
further explored [58]. We speculate that maternal aging
creates a suboptimal environment for placental and fetal
development that contributes to the vulnerability to
adverse outcomes.
In this population adverse pregnancy outcome was
associated with higher circulating levels of 8-isoprostane
(See figure on next page.)
Fig. 5  Levels of placental hormones in maternal serum from women ≥ 40 years of age with normal and adverse pregnancy outcome (n = 43/
group). Maternal serum/plasma at 28 weeks (A, C, E, G) and 36 weeks (B, D, F, H) gestation against pregnancy outcome. A, B human placental
lactogen (hPL), C, D Placental growth factor (PlGF), E, F Soluble fms like tyrosine kinase-1 (sFlt) and G, H PlFG:sFlt ratio. Data presented as median,
interquartile range (box) and total range (whiskers) and analysed using Mann-Whitney U-test (*p < 0.05, **p < 0.01, ***p < 0.005)
Page 13 of 17
at 28 weeks’ gestation, indicating elevated oxidative dam-
age, despite higher antioxidant capacity. Inadequate
antioxidant compensatory responses resulting in oxida-
tive stress has been detected in placentas from adverse
pregnancy outcome, where it has been related to altered
placental function [22]. Future studies are required to
confirm whether placental oxidative damage is impli-
cated in the placental dysfunction observed in pregnan-
cies in older mothers.
Consistent with other reports reduced maternal circu-
lating concentrations of placental hormones (hPL, sFlt
and PlGF) were detected in pregnancies in older women
with adverse outcome compared to normal outcomes.
The differences in placental hormones provide further
evidence for placental dysfunction as an underpinning
mechanism for susceptibility to adverse outcomes in
older women. There is a growing body of evidence that
these represent biomarkers of placental dysfunction, and
are lower in pregnancies with FGR, PE and stillbirth [59–
61]. PlGF and sFlt concentrations in the maternal circu-
lation are correlated with fetal size as early as the first
trimester [62] and have strong potential as biomarkers for
adverse pregnancy outcome in a clinical setting [61, 63].
In common with prognostic accuracy studies from other
contexts in pregnancy, PlGF had the strongest predictive
value for adverse pregnancy outcome in older mothers
[64], although the AUROC is insufficient for clinical util-
ity at present. Further studies with larger sample size are
required to determine if a prognostic model incorporat-
ing the clinical and biochemical predictors with sufficient
predictive power to identify older women at greatest risk
of adverse outcome can be derived.
Conclusion
This study has identified alterations in circulating bio-
markers of inflammation and oxidative stress markers in
older pregnant women, suggesting that biological pro-
cesses seen in aging may contribute to susceptibility to
adverse outcomes in this population. Furthermore, we
identified serum biomarkers with fair predictive accu-
racy for adverse pregnancy outcome in older women.
With larger sample sizes and data sets, and by combining
identified demographic and clinical variables that alter
risk of adverse outcome and the measurement of aging
and placental biomarkers, it may be possible to create a
Lean et al. BMC Pregnancy Childbirth (2021) 21:706 Page 14 of 17

Fig. 5  (See legend on previous page.)

 Lean et al. BMC Pregnancy Childbirth (2021) 21:706
Fig. 6  Predictive values of biomarkers of adverse pregnancy outcome in women of advanced maternal age. ROC curve of (A) TAC (Area under
the ROC curve (AUROC) =0.69), (B) 8-Isoprostane (AUROC = 0.66), (C) hPL (AUROC = 0.68) and (D) PlGF (AUROC = 0.74) as predictors of adverse
pregnancy outcome
predictive model with sufficient discrimination to delin-
eate an individual woman’s risk of adverse pregnancy
outcome relating to advanced maternal age. This would
allow older mothers to be offered more individualised
care that considers both maternal and fetal wellbeing and
reduces stillbirth rates whilst minimising unnecessary
intervention.
Abbreviations
AOR: Adjusted odds ratio; ART​: Assisted reproductive technology; AUROC:
Area under the receiver operator characteristic curve; BMI: Body mass index;
ELISA: Enzyme linked immunosorbent assay; FGR: Fetal growth restriction; hPL:
Human placental lactogen; IBC: Individualised birthweight centile; IL: Inter-
leukin; IMD: Index of multiple deprivation; IOL: Induction of labour; MAMAS:
Manchester Advanced Maternal Age Study; NICU: Neonatal intensive care unit;
OR: Odds ratio; PE: Preeclampsia; PlGF: Placental growth factor; PTB: Preterm
birth; sFlt1: Soluble fms-like tyrosine kinase 1; TNF: Tumour necrosis factor.
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12884-​021-​04178-6.
Additional file 1.
Acknowledgements
We would like to thank the participating hospitals in MAMAS study, namely;
University Hospital of North Staffordshire: City General Site, Stoke-on-Trent;
Leighton Hospital: Women’s Health Unit, Crewe; South Warwickshire Founda-
tion NHS Trust, Warwick; Burton Hospitals NHS Foundation Trust: Queens
Hospital Burton, Burton-on-Trent; Lincoln County: Clinical Research Facility,
Page 15 of 17
Lincoln; and Pilgrim Hospital, Boston. We acknowledge the primary investiga-
tors at these sites: Dr. Peter Young, Miss Karren McIntyre, Dr. Nicole Pope, Mr.
Olanreqaju Sorinola, Mr. Odiri Oteri and Mr. Sunday Ikhena for overseeing
MAMAS across the UK. Strong thanks must be given to Linda Peacock and
Jane Boscolo-Ryan, the Research Midwives in the primary MAMAS site, St.
Mary’s Hospital, Manchester, who were pivotal in the initiation and progres-
sion of MAMAS. Furthermore, we would like to acknowledge the hard work of
all the research midwives without whom this study would never have been
such a success. Finally, we would like to thank all the mothers who kindly
agreed to participate in MAMAS.
Authors’ contributions
Contribution of Authorship: Conception (AEPH, RLJ, SCL), planning (AEPH, RLJ,
SCL), carrying out (SCL), analysing (SCL, SAR, AEPH, RLJ, SCL, SAR), writing up
(SCL, RLJ, AEPH). The author(s) read and approved the final manuscript.
Funding
This study was funded by Tommy’s Charity. The funding source had no
involvement in study design, manuscript preparation or decision to publish.
Availability of data and materials
The datasets generated and/or analysed during the current study are not
publicly available as ethnical approval was not sought for their dissemination
but are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This study was given a favourable ethical opinion by Greater Manchester
South Research Ethics Committee (Ref 12/NW/0015). All participants gave
written informed consent prior to participation; all methods were performed
in accordance with the relevant guidelines and regulations.
Competing interests
The authors declare that they have no competing interests.
Lean et al. BMC Pregnancy Childbirth (2021) 21:706
Author details
1
 Maternal and Fetal Health Research Centre, Division of Developmental Biol-
ogy and Medicine, Faculty of Biology, Medicine and Health, University of Man-
chester, St. Mary’s Hospital, 5th Floor (Research), Oxford Road, Manchester M13
9WL, UK. 2 Centre for Biostatistics, Faculty of Biology, Medicine and Health,
University of Manchester, Manchester, UK.
Received: 16 December 2020 Accepted: 28 September 2021
References
1. Office of National Statistics. Births by parents’ characteristics 1938–2016.
London: Office of National Statistics; 2018.
2. Royal College Of Obstetricians and Gynaecologists. Induction of Labour
at Term in Older Mothers Scientific Impact Paper No. 34. London: Royal
College of Obstetricians and Gynaecologists; 2013.
3. Bahtiyar M, Funai E, Norwitz E, Buhimschi C, Rosenberg V, Copel J.
Advanced maternal age (AMA) is an independent predictor of intrauter-
ine fetal death at term. Am J Obstet Gynecol. 2006;195:S209.
4. Carolan M. Maternal age >/=45 years and maternal and perinatal out-
comes: a review of the evidence. Midwifery. 2013;29(5):479–89.
5. Cleary-Goldman J, Malone FD, Vidaver J, Ball RH, Nyberg DA, Comstock
CH, et al. Impact of maternal age on obstetric outcome. Obstet Gynecol.
2005;105(5 Pt 1):983–90.
6. Jahromi BN, Husseini Z. Pregnancy outcome at maternal age 40 and
older. Taiwan J Obstet Gynecol. 2008;47(3):318–21.
7. Kenny LC, Lavender T, McNamee R, O’Neill SM, Mills T, Khashan AS.
Advanced maternal age and adverse pregnancy outcome: evidence from
a large contemporary cohort. PLoS One. 2013;8(2):e56583.
8. Lean SC, Derricott H, Jones RL, Heazell AEP. Advanced maternal age and
adverse pregnancy outcomes: a systematic review and meta-analysis.
PLoS One. 2017;12(10):e0186287.
9. Fretts RC. Etiology and prevention of stillbirth. Am J Obstet Gynecol.
2005;193(6):1923–35.
10. Flenady V, Koopmans L, Middleton P, Froen JF, Smith GC, Gibbons K, et al.
Major risk factors for stillbirth in high-income countries: a systematic
review and meta-analysis. Lancet. 2011;377(9774):1331–40.
11. Waldenstrom U, Aasheim V, Nilsen AB, Rasmussen S, Pettersson HJ,
Schytt E. Adverse pregnancy outcomes related to advanced maternal
age compared with smoking and being overweight. Obstet Gynecol.
2014;123(1):104–12.
12. Royal College Of Obstetricians and Gynaecologists. Care of Women with
Obesity in Pregnancy (Green-top Guideline No. 72). London: RCOG; 2018.
13. Excellence NIfHaC. Smoking: stopping in pregnancy and after childbirth
(PH26) 2010 [Available from: https://​www.​nhs.​uk/​condi​tions/​pregn​ancy-​
and-​baby/​foods-​to-​avoid-​pregn​ant/.
14. Fretts RC, Duru UA. New indications for antepartum testing: making
the case for antepartum surveillance or timed delivery for women of
advanced maternal age. Semin Perinatol. 2008;32(4):312–7.
15. Johnson JA, Tough S, Sogc GC. Delayed child-bearing. J Obstet Gynaecol
Can. 2012;34(1):80–93.
16. Walker KF, Bugg G, Macpherson M, McCormick C, Wildsmith C, Smith G,
et al. Induction of labour versus expectant management for nulliparous
women over 35 years of age: a multi-Centre prospective, randomised
controlled trial. BMC Pregnancy Childbirth. 2012;12:145.
17. Walker KF, Bugg GJ, Macpherson M, Thornton J. Induction of labour at
term for women over 35 years old: a survey of the views of women and
obstetricians. Eur J Obstet Gynecol Reprod Biol. 2012;162(2):144–8.
18. Bahtiyar MO, Funai EF, Rosenberg V, Norwitz E, Lipkind H, Buhimschi C,
et al. Stillbirth at term in women of advanced maternal age in the United
States: when could the antenatal testing be initiated? Am J Perinatol.
2008;25(5):301–4.
19. Walker KF, Bugg GJ, Macpherson M, McCormick C, Grace N, Wildsmith C,
et al. Randomized trial of labor induction in women 35 years of age or
older. N Engl J Med. 2016;374(9):813–22.
20. Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen
species, oxidative stress and antioxidants in pregnancy outcomes. Int J
Biochem Cell Biol. 2010;42(10):1634–50.
Page 16 of 17
21. Mullins E, Prior T, Roberts I, Kumar S. Changes in the fetal and neonatal
cytokine profile in pregnancies complicated by fetal growth restriction.
Am J Reprod Immunol. 2013;69(5):441–8.
22. Myatt L, Cui X. Oxidative stress in the placenta. Histochem Cell Biol.
2004;122(4):369–82.
23. Schetter AJ, Heegaard NH, Harris CC. Inflammation and cancer: inter-
weaving microRNA, free radical, cytokine and p53 pathways. Carcinogen-
esis. 2010;31(1):37–49.
24. Barja G. The mitochondrial free radical theory of aging. Prog Mol Biol
Transl Sci. 2014;127:1–27.
25. de Steenwinkel FD, Hokken-Koelega AC, de Man YA, de Rijke YB, de
Ridder MA, Hazes JM, et al. Circulating maternal cytokines influence fetal
growth in pregnant women with rheumatoid arthritis. Ann Rheum Dis.
2013;72(12):1995–2001.
26. Myatt L. Review: reactive oxygen and nitrogen species and functional
adaptation of the placenta. Placenta. 2010;31(Suppl):S66–9.
27. Girard S, Heazell AE, Derricott H, Allan SM, Sibley CP, Abrahams VM, et al.
Circulating cytokines and alarmins associated with placental inflamma-
tion in high-risk pregnancies. Am J Reprod Immunol. 2014;72(4):422–34.
28. Lean SC, Heazell AEP, Dilworth MR, Mills TA, Jones RL. Placental dysfunc-
tion underlies increased risk of fetal growth restriction and stillbirth in
advanced maternal age women. Sci Rep. 2017;7(1):9677.
29. Jordan H, Roderick P, Martin D. The index of multiple deprivation 2000
and accessibility effects on health. J Epidemiol Community Health.
2004;58(3):250–7.
30. Gardosi J, Francis A. GROW. In: Customised Weight Centile Calculator.
6.7.7 ed. Birmingham: Gestational Network; 2015.
31. Alberti-Fidanza A, Di Renzo GC, Burini G, Antonelli G, Perriello G. Diet dur-
ing pregnancy and total antioxidant capacity in maternal and umbilical
cord blood. J Matern Fetal Neonatal Med. 2002;12(1):59–63.
32. Musolino C, Alonci A, Allegra A, Saija A, Penna G, Cannavo A, et al.
Increase in serum protein carbonyl groups is associated with more
advanced stage of disease in multiple myeloma patients. Biomarkers.
2011;16(8):718–9.
33. Ogawa F, Shimizu K, Muroi E, Hara T, Hasegawa M, Takehara K, et al.
Serum levels of 8-isoprostane, a marker of oxidative stress, are
elevated in patients with systemic sclerosis. Rheumatology (Oxford).
2006;45(7):815–8.
34. Kikuchi A, Takeda A, Onodera H, Kimpara T, Hisanaga K, Sato N, et al.
Systemic increase of oxidative nucleic acid damage in Parkinson’s disease
and multiple system atrophy. Neurobiol Dis. 2002;9(2):244–8.
35. Bartha JL, Romero-Carmona R, Comino-Delgado R. Inflammatory
cytokines in intrauterine growth retardation. Acta Obstet Gynecol Scand.
2003;82(12):1099–102.
36. Doria A, Cutolo M, Ghirardello A, Zen M, Villalta D, Tincani A, et al. Effect
of pregnancy on serum cytokines in SLE patients. Arthritis Res Ther.
2012;14(2):R66.
37. Tosun M, Celik H, Avci B, Yavuz E, Alper T, Malatyalioglu E. Maternal and
umbilical serum levels of interleukin-6, interleukin-8, and tumor necrosis
factor-alpha in normal pregnancies and in pregnancies complicated by
preeclampsia. J Matern Fetal Neonatal Med. 2010;23(8):880–6.
38. Stacey T, Thompson JM, Mitchell EA, Ekeroma AJ, Zuccollo JM, McCowan
LM. Relationship between obesity, ethnicity and risk of late stillbirth: a
case control study. BMC Pregnancy Childbirth. 2011;11:3.
39. Heazell A, Budd J, Smith LK, Li M, Cronin R, Bradford B, et al. Associations
between social and behavioural factors and the risk of late stillbirth - find-
ings from the Midland and North of England Stillbirth case-control study.
BJOG. 2021;128(4):704-13.
40. Draper ES, Gallimore ID, Kurinczuk JJ, Smith PW, Boby T, Smith LK, et al.
MBRRACE-UK Perinatal Mortality Surveillance Report, UK Perinatal Deaths
for Births from January to December 2016. Leicester: The Infant Mortality
and Morbidity Studies, Department of Health Sciences, University of
Leicester; 2018.
41. Alio AP, Salihu HM, McIntosh C, August EM, Weldeselasse H, Sanchez
E, et al. The effect of paternal age on fetal birth outcomes. Am J Mens
Health. 2012;6(5):427–35.
42. Astolfi P, De Pasquale A, Zonta LA. Late paternity and stillbirth risk. Hum
Reprod. 2004;19(11):2497–501.
43. Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS. Low and
very low birth weight in infants conceived with use of assisted reproduc-
tive technology. N Engl J Med. 2002;346(10):731–7.
 Lean et al. BMC Pregnancy Childbirth (2021) 21:706
44. Luke B, Stern JE, Kotelchuck M, Declercq ER, Cohen B, Diop H. Birth out-
comes by infertility diagnosis analyses of the Massachusetts outcomes
study of assisted reproductive technologies (MOSART). J Reprod Med.
2015;60(11–12):480–90.
45. Myrskyla M, Barclay K, Goisis A. Advantages of later motherhood. Gynako-
loge. 2017;50(10):767–72.
46. Heazell A, Budd J, Smith LK, Li M, Cronin R, Bradford B, et al. Associations
between social and behavioural factors and the risk of late stillbirth - find-
ings from the Midland and north of England stillbirth case-control study.
BJOG. 2021;128(4):704–13.
47. Syslova K, Bohmova A, Mikoska M, Kuzma M, Pelclova D, Kacer P. Multi-
marker screening of oxidative stress in aging. Oxidative Med Cell Longev.
2014;2014:562860.
48. Checa J, Aran JM. Reactive oxygen species: drivers of physiological and
pathological processes. J Inflamm Res. 2020;13:1057–73.
49. Miossec P. Pro- and antiinflammatory cytokine balance in rheumatoid
arthritis. Clin Exp Rheumatol. 1995;13(Suppl 12):S13–6.
50. Sharps MC, Baker BC, Guevara T, Bischof H, Jones RL, Greenwood SL, et al.
Increased placental macrophages and a pro-inflammatory profile in pla-
centas and maternal serum in infants with a decreased growth rate in the
third trimester of pregnancy. Am J Reprod Immunol. 2020;84(3):e13267.
51. Burton GJ, Jauniaux E. Pathophysiology of placental-derived fetal growth
restriction. Am J Obstet Gynecol. 2018;218(2S):S745–S61.
52. Tenorio MB, Ferreira RC, Moura FA, Bueno NB, de Oliveira ACM, Goulart
MOF. Cross-talk between oxidative stress and inflammation in preeclamp-
sia. Oxidative Med Cell Longev. 2019;2019:8238727.
53. Kaya S, Keskin HL, Kaya B, Ustuner I, Avsar AF. Reduced total antioxidant
status in postterm pregnancies. Hippokratia. 2013;17(1):55–9.
54. Amu S, Hahn-Zoric M, Malik A, Ashraf R, Zaman S, Kjellmer I, et al.
Cytokines in the placenta of Pakistani newborns with and without intrau-
terine growth retardation. Pediatr Res. 2006;59(2):254–8.
55. Thaxton JE, Romero R, Sharma S. TLR9 activation coupled to IL-10
deficiency induces adverse pregnancy outcomes. J Immunol.
2009;183(2):1144–54.
56. Chatterjee P, Chiasson VL, Kopriva SE, Young KJ, Chatterjee V, Jones KA,
et al. Interleukin 10 deficiency exacerbates toll-like receptor 3-induced
preeclampsia-like symptoms in mice. Hypertension. 2011;58(3):489–96.
Page 17 of 17
57. Lai Z, Kalkunte S, Sharma S. A critical role of interleukin-10 in modulat-
ing hypoxia-induced preeclampsia-like disease in mice. Hypertension.
2011;57(3):505–14.
58. Da Silva EC, Silva SV, Damiao R, Fonseca EB, Garcia S, Lippi UG. Stress
and anxiety in pregnant women exposed to ultrasound. J Matern Fetal
Neonatal Med. 2012;25(3):295-8.
59. Ranta JK, Raatikainen K, Romppanen J, Pulkki K, Heinonen S. Decreased
PAPP-A is associated with preeclampsia, premature delivery and small
for gestational age infants but not with placental abruption. Eur J Obstet
Gynecol Reprod Biol. 2011;157(1):48–52.
60. Dutton PJ, Warrander LK, Roberts SA, Bernatavicius G, Byrd LM, Gaze D,
et al. Predictors of poor perinatal outcome following maternal perception
of reduced fetal movements--a prospective cohort study. PLoS One.
2012;7(7):e39784.
61. Kenny LC, Black MA, Poston L, Taylor R, Myers JE, Baker PN, et al. Early
pregnancy prediction of preeclampsia in nulliparous women, combin-
ing clinical risk and biomarkers: the screening for pregnancy endpoints
(SCOPE) international cohort study. Hypertension. 2014;64(3):644–52.
62. Bouwland-Both MI, Steegers EA, Lindemans J, Russcher H, Hofman A,
Geurts-Moespot AJ, et al. Maternal soluble fms-like tyrosine kinase-1,
placental growth factor, plasminogen activator inhibitor-2, and folate
concentrations and early fetal size: the Generation R study. Am J Obstet
Gynecol. 2013;209(2):121 e1–11.
63. Herraiz I, Simon E, Gomez-Arriaga PI, Martinez-Moratalla JM, Garcia-Bur-
guillo A, Jimenez EA, et al. Angiogenesis-related biomarkers (sFlt-1/PLGF)
in the prediction and diagnosis of placental dysfunction: an approach for
clinical integration. Int J Mol Sci. 2015;16(8):19009–26.
64. Heazell AE, Hayes DJ, Whitworth M, Takwoingi Y, Bayliss SE, Davenport
C. Biochemical tests of placental function versus ultrasound assessment
of fetal size for stillbirth and small-for-gestational-age infants. Cochrane
Database Syst Rev. 2019;5:CD012245.
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